Variant Creutzfeldt-Jakob disease (vCJD) is a rare and fatal human neurodegenerative condition. As with Creutzfeldt-Jakob disease, vCJD is classified as a transmissible spongiform encephalopathy (TSE) because of characteristic spongy degeneration of the brain and its ability to be transmitted. vCJD is a new disease that was first described in March, 1996. In contrast to the traditional forms of CJD, vCJD has affected younger patients (average age 29 years, as opposed to 65 years), has a relatively longer duration of illness (median of 14 months as opposed to 4.5 months) and is strongly linked to exposure, probably through food, to a TSE of cattle called Bovine spongiform encephalopathy (BSE). TSEs are also known in other animals. For instance, scrapie affects sheep and goats and has been found in many sheep-producing countries throughout the world for over 250 years. Chronic wasting disease (CWD) is a contagious fatal TSE in cervids (members of the deer and elk family).
The hypothesis of a link between vCJD and BSE was first raised because of the association of these two TSEs in time and place. More recent evidence supporting a link includes identification of pathological features similar to vCJD in brains of macaque monkeys inoculated with BSE. A vCJD-BSE link is further supported by the demonstration that vCJD is associated with a molecular marker that distinguishes it from other forms of CJD and which resembles that seen in BSE transmitted to a number of other species. Studies of the distribution of the infectious agent in the brains of mice artificially infected with tissues from humans with vCJD and cows with BSE showed nearly identical patterns. The most recent and powerful evidence comes from studies showing that the transmission characteristics of BSE and vCJD in laboratory mice are almost identical, strongly indicating that they are due to the same causative agent. In conclusion, the most likely cause of vCJD is exposure to the BSE agent, most plausibly due to dietary contamination by affected bovine central nervous system tissue.
The infectious agents involved in TSE are usually referred to as prions. These pathogens are characterized by unusual properties and, in particular, by their strong resistance to common procedures of disinfection used against conventional microorganisms. Prions that cause BSE in cattle and vCJD in humans are transmitted through infected food products. While the incubation period of the disease is quite long, the onset of symptoms leads to extreme neurological debilitation within a few months. All animals possess normal prion proteins, which are known by a variety of acronyms in the scientific literature, e.g., PrP or PrPc. PrPc are found on the surface of many cell types including nerves, lymphocytes, and macrophages. All proteins “fold” themselves into specific three-dimensional shapes. Sometimes normal prion proteins become abnormally folded. Then the abnormal prions can “infect” normal ones by physical contact in which the abnormally-folded prion causes the normal one also to become misfolded, thus spreading the diseased state.
A major component of highly infectious prion protein fractions is a Proteinase K-resistant prion protein, termed PrPsc. The normal host prion protein PrPc is sensitive to Proteinase K. The biochemical behavior of PrPsc under denaturing conditions and in the presence of Proteinase K provides a biochemical means for assaying the presence of PrPsc in tissue samples, thereby diagnosing TSE disease. Identification of infected farm animals such as cattle or sheep post mortem is of particular importance in order to prevent potentially hazardous meat and other products from entering the human food chain. As the number of animals slaughtered and processed by the meat industry is high, TSE assays which are to be performed by foodstuff monitoring centers and reference laboratories have to meet the needs of high throughput testing.
A general workflow to assay for TSE essentially comprises the following steps:    (1) Removing sample tissue from a sacrificed animal            In the exemplary case of BSE the sample is preferably a piece of brain; usually, a part of the brain stem is removed when the animal is cut up. Preferably, the piece of brain includes the obex. However, other tissues are possible including spinal chord tissue and also lymph node or tonsil tissue.            (2) Taking a defined amount of tissue, which in the following will be referred to as “the sample”, “the sampled tissue”, or “the tissue sample”    (3) Homogenizing th tissue sample    (4) Treating the homogenized sample such that it is suitable for specific detection of PrPsc, if present            A preferred pretreatment uses proteolytic digestion of the homogenized sample with Proteinase K and denaturation with a chaotropic agent.            (5) Incubating the treated sample with one or more specific binding agents            Usually the binding agent is an antibody or an antibody fragment. The amount of complex formed by the binding agent(s) and the analyte is measured to result in a measurement value. In case a sample treated with Proteinase K and chaotropic agent is analyzed, the binding agents are specific for a fragment of PrPsc which resists the Proteinase K treatment.            (6) In order to assign a quantitative value to a positive or negative test result, using an algorithm which takes into account reference data and/or measurement values generated in one or more control experiments
The present invention focuses on steps (3), (4), (5) and (6) of the workflow. Step (5) according to the invention includes pretreatment with a proteolytic enzyme and chaotropic agent.
With respect to commercialized test systems, there exist several ways of performing steps (3) to (6). Generally, the processing of infective tissue often requires complex equipment in order to fulfill the needs of laboratory safety on the one hand and technical requirements on the other.
A first test system developed by Prionics AG (Schlieren, Switzerland) is distributed under the commercial name PRIONICS-Check WESTERN and PRIONICS-Check LIA (=luminescence immunoassay, described further below). The PRIONICS-Check LIA test system comprises (i) homogenization of sample tissue using a complex device and apparatus, (ii) proteolytic sample treatment, and (iii) immunological detection of PrPsc, whereby steps (ii) and (iii) are performed in the 96-well format.
According to the user's manual of the test system, for the homogenization step a deconstituting device for the preparation of biological samples as described in WO 02/48679 is used. The device comprises a container in the form of a cup for holding the tissue to be deconstituted. A shaft is mounted for rotation inside the container with a blade on the end inside the container. The shaft is supported axially by ball coupling means and has engagement means on its end outside the container for coupling the shaft to a motor. The device is used in an automatic apparatus (FASTH PCPM4, cod. 80040, Consul A R, Villeneuve, Switzerland), which includes at least one support element with a plurality of housings for receiving the container; a deconstitution station which includes at least one motor with a drive shaft for engaging the engagement means of the shaft of the deconstituting device, whereby the motor is movable between a position disengaged from the shaft and one position engaging it; and conveyor means for transporting the support element to the deconstitution station. The FASTH device simultaneously processes 4 containers which are placed in a rack. The rack takes up eight containers which are processed in two subsequent steps. Another device (MEDIFASTH, Consul) is commercially available which works similarly but processes only two containers at a time. The containers are placed in racks, whereby each rack takes up two containers.
The containers are commercially available as disposable items (PRYPCONS, Consul A R, Villeneuve, Switzerland) made of plastic. Each container has a sliding gate in the lid through which the sampled brain tissue is transferred into the container. The FASTH device is capable of taking up and subsequently processing 6 racks, i.e., 48 containers. Racks with processed containers can be removed from the running device; similarly, the device can be loaded with new racks. According to the manufacturer the device is capable of homogenizing up to 250 samples/h.
In each container a sample of 350-700 mg of tissue is homogenized together with a ten-fold volume of homogenization buffer. In order to ensure a proper sample vs. buffer ratio, the weight of the sample tissue is determined before. Following the homogenization process, an aliquot of the homogenate (1 ml) is taken manually from the container, usually with a hand pipet using disposable pipet tips, and is transferred to a 96-well “sample master plate”. Alternatively, the homogenate can be removed and transferred by means of an automatic pipetting device (e.g., supplied by TECAN Group Ltd., Switzerland). However, the sliding gate of each container has to be opened manually.
In a cavity of a microwell plate (the “digestion” plate), a volume of 100 μl of the homogenate is mixed with 50 μl of a digestion solution containing a protease. The mixture is then sealed and incubated. Following an incubation, 10 μl of a stop solution are added to the digested homogenate and mixed. A volume of 15 μl of the mixture is transferred to a cavity of another microwell plate (the “preincubation plate”) which contains 15 μl of an “assay buffer”. Transfer and mixing of samples with assay buffer have to be completed within 2-5 min. After 2-5 min a volume of 10 μl “preincubation buffer” is added, mixed, and incubated for about 2 min. Subsequently, 200 μl of “detection antibody solution” are added and mixed. The plate is sealed and incubated on a shaker for about 60 min. Subsequently, a volume of 200 μl of each cavity is transferred to the corresponding cavity of a “capture plate”. The plate is then incubated for about 90 min on a shaker. After that, the capture plate is washed with wash buffer four times using an ELISA washer. Remaining liquid is removed (preferably by clapping), and each cavity is filled with 100 μl of a chemiluminescent substrate working solution. After an incubation period of 5-10 min, the light signals are read in a chemiluminometer, and the signals are recorded.
A different test system is the Enfer TSE diagnostic kit which is distributed by Abbott Laboratories, USA. The homogenization method depends on the Stomacher apparatus (Seward Stomacher 80). The apparatus provides a mechanical action in which contoured paddles apply pressure to a sample bag. Sample tissue in the sample bag is subjected to homogenization in the presence of a sample buffer. This method is suited for soft tissue such as obex. A single apparatus is capable of processing 2 bags at a time. Homogenization of a tissue sample typically takes 2 min. For each single sample preparation a new blade, tongue depressor, weighing boat, and homogenizer bag must be used to prevent cross-contamination. The tissue sample is a thin cross-section of CNS tissue weighing between at least 0.5 g and 1.0 g. Weighing and transferring the sample into the bag require extensive manual work. Per 1 g of tissue, 15 ml of sample buffer are added into the bag. When dispensing the sample buffer, care must be taken to avoid contact of the dispenser with the bag. Homogenization is controlled by visual inspection. After the homogenization step, the homogenizer bag is left between 5 min and 40 min to allow bubbles to subside.
Following the homogenization process, the homogenization bag is opened and an aliquot (180 μl) of the liquid homogenate is taken manually out of the bag with a hand pipet using disposable pipet tips. At this point care must be taken not to allow the pipette to be contaminated on the outside by material from the homogenizer bag. The homogenate is transferred to a 96-well microwell plate. The plate is sealed and subjected to centrifugation at 4,000×g at room temperature for 5 min. One hundred μl of the supernatant are then transferred to a second 96-well microwell plate containing 20 μl of a Proteinase K solution. According to the user's manual, the Proteinase K solution has a tendency to stick to the side of the cavities. In order to prevent this, the buffer has to be pipetted to the very bottom of the cells. When transferring the supernatant, care must be taken not to disturb the pellet because transfer of solid particles must be prevented. The plate is then covered with a plate sealer, and samples are then incubated for 60 min on a shaker. During this step, PrPsc aggregates are hydrophobically bound to the cavities of the microwell plate while the Proteinase K digests away the PrPc, thus allowing the distinction of PrPsc from PrPc. The pipetting steps of this protocol can be automated.
The cavities of the microwell plate are washed, and the plate is clapped to remove remaining liquid. Subsequently, the sample cavities are incubated with 150 μl of Enfer buffer 3 for 15 min. The plate is washed again, clapped, and after that, rabbit polyclonal antibody specific for the PrP protein in a volume of 150 μl is added to the cavities. The plate is sealed again and incubated for 40 min. After that, the cavities are washed again and then incubated with an enzyme-conjugated second antibody. After incubation with the conjugate, the cavities are washed, and chemiluminescent substrate is added. The light signal is read in a chemiluminometer and the signal is recorded.
Another commercially available test system is the TeSeE BSE test distributed by Bio-Rad Laboratories (Munich, Germany; catalogue numbers 3551144 [purification kit], 3551145 [detection kit], 3551120 [calibration syringe and needle]). This method is adapted for processing two times 45 samples (plus controls) at a time, whereby in the beginning test tubes in racks in the 6*8 format are used.
An amount of 350 μg±40 μg of brain stem tissue, preferred obex tissue, is transferred to a grinding tube. This is a test vial with a volume of about 2 ml containing about 1.5 ml homogenization buffer and grinding beads. Test vials pre-filled with beads and buffer are part of the Bio-Rad kit. The grinding beads have a diameter of between 0.5 mm and 1 mm. The tube is filled with grinding beads up to about the 200 μl marking. Upon transfer of sample tissue, each test vial is closed manually with a screw cap, and the vial is placed in a rack which holds 48 vials in total. The homogenization step is performed by using a RIBOLYSER or a TeSeE Precess 48 device. Basically, the device agitates the racks with the vials, and the movement of the beads in each vial homogenizes the sample tissue contained therein.
Following the homogenization step, the rack is removed from the agitation device, each vial is opened manually, and a volume of 250 μl of the homogenate is removed using a syringe for single use which is fitted with a calibrated blunt end needle. The needle needs to be immersed in the pellet of beads to avoid taking up poorly homogenized tissue fragments.
The homogenates are transferred to reaction vials for further treatment. An equal volume of Proteinase K solution is added, mixed, and incubated at 37° C. for 10 min. Mixing is done manually by inverting the tubes 10 times. According to the user's manual, the time between adding Proteinase K solution and the incubation at 37° C. must not exceed 2 min. After the incubation, a volume of 250 μl of a “clarifying solution” (reagent B) is added and mixed. It is noted that for this step, the Eppendorf vial has to be manually opened and closed again. It is also noted that according to the user's manual, the time after the incubation until the point when the clarifying solution is mixed must not exceed 2 min. Within 30 min, the Eppendorf reaction vials are centrifuged for 5 min at 20,000×g. The vials are opened again, and the supernatants are discarded. The vials are dried by inverting onto absorbent paper for 5 min. Subsequently, a volume of 25 μl of “resolving buffer” (reagent C) is added to each vial, the vial is closed again and then immediately incubated at 100° C. for 5 min. After that, in another manual step, the vials are agitated on a vortex mixer for 5 seconds.
The treated sample is then diluted with 125 μl PBS containing BSA and mixed by vortexing just before transfer to a microwell plate. One hundred μl of the diluted sample are transferred to the microwell plate which is coated with a monoclonal capture antibody. The plate is sealed with adhesive tape. After incubation of the plate for about 75 min at 37° C., the plate is washed using an automatic washer, and subsequently 100 μl of a solution containing an enzyme-conjugated detection antibody are added to each cavity. The plate is sealed again with adhesive tape. After incubation of the plate for about 1 h, the plate is washed again, and a volume of 100 μl of a substrate solution is added to each cavity. After 30 min, the enzyme reaction is stopped by adding a stop solution. Signals are read in a microwell plate reader, and the signals are recorded.
The methods of the state of the art have certain disadvantages. Particularly the homogenization process represents a bottleneck with regard to both susceptibility to contamination and sample throughput. It is noted that a test system which would be performed entirely in the standardized 8×12 (i.e., 96-well) format would be advantageous. Also, a cost-effective workflow in this format with reduced hands-on time is desired.
For the PRYPCON container it is noted that the tissue is homogenized in the container in the presence of a homogenization buffer by means of fast rotating (20,000 r.p.m.) blades. As a consequence, an aerosol containing components of possibly prion-infected brain tissue is generated in the airspace in the inner compartment of the container. Regarding the container it is noted in addition that both (i) the opening of the lid with the shaft inserted and (ii) the sliding gate do not provide completely airtight (i.e., pressure-tight) sealing of the inner compartment where the homogenization takes place. The sample bags used for homogenization with the Stomacher apparatus may also give rise to contamination, particularly when the homogenate is removed from the bag.
Regarding throughput, the FASTH device is capable of simultaneously processing 4 PRYPCON containers. While this system already provides a partly automated homogenization process, the alternative system using Stomacher bags is capable of processing only 2 bags at a time per device. The Bio-Rad system, in contrast, homogenizes 48 samples at a time.
However, during the sample preparation procedure of the Bio-Rad system, each grinding tube is closed manually with a screw cap. Twisting of the screw cap presses a seal onto the mouth of the container; the stronger the screw cap is twisted, the tighter the tube is sealed. That is to say, depending on the force exerted when twisting the cap to close the vial, the sealing effect of the screw cap may be imperfect. As a consequence of imperfect sealing, liquid contents of the vial may be released from a screw-capped vial, e.g., in case the pressure inside the vial rises relative to the outside due to a rise of temperature in the vial or a drop of air pressure outside. Thus, the use of screw caps poses a problem when “sealing” as a defined (i.e., standardized) state or result is to be obtained. An exemplary parameter for standardized sealing is air-tightness over a defined time interval against a measured relative difference of higher air pressure within the container and lower air pressure outside of the sealed container.
In addition, the use of screw caps requires increased manual handling by lab technicians and prevents rapid processing. Consequently, screw caps pose a problem when larger number of vials have to be opened and closed, or when it is desired to seal the vials automatically. In addition, the more manual handling is involved, the higher are the chances that vials with sample material are mistakenly interchanged.
The amount and the complexity of the material for single use which is needed to perform a TSE assay influence the price of an assay system. However, the amount of single use items and the material of which these are made particularly impact on the costs of waste disposal. Depending on national regulations, any waste material which has come in contact with infective TSE agent has to be subjected to chemical and/or heat treatment, autoclaving, or incineration. Due to the difficulties to inactivate infective prions, these processes are more cost-intensive compared to the inactivation of usual infective waste. Therefore, it is desired to minimize expenditure on waste disposal when running TSE tests at high throughput.